The ability to precisely manipulate individual molecules has led to stunning new discoveries in physics, biology, and medicine (Deniz A. A. et al. (2008) J. R. Soc. Interface, 5: 15; Ritort F. et al. (2006) J. Phys.: Condens. Matter 18: R531), as well as powerful new methods in nanoscale engineering. For example, single-molecule force measurements have revealed the basic mechanical properties of nucleic acids (Bustamante C. et al. Nature 421: 423-7), the dynamics and functioning of molecular motors (Svoboda K. et al. (1993) Nature 365: 721-7; Greenleaf W. J. et al. (2007) Annu. Rev. Biophys. Biomol. Struct. 36: 171), and the role of hydrodynamic forces in the circulatory system in regulating enzymatic activity (Zhang X. et al. (2009) Science 324: 1330). In addition, these measurements have yielded fundamental insights into the dynamical strength of molecular interactions (Evans E. (2001) Annu. Rev. Biophys. Biomol. Struct. 30: 105-28), which have led to the development of creative new tools for nanoscale assembly (Kufer S. K. et al. (2008) Science 319: 594).
Mechanical forces can be applied to individual molecules using a broad range of tools, including optical traps, magnetic tweezers, mechanical cantilevers, and the centrifuge force microscope (Neuman K. C. et al. (2008) Nature Methods 5: 491-505; Halvorsen K. et al. (2010) Biophys. J. 98: L53-5). Yet a common requirement of these methods is that single-molecule constructs must be specifically tethered between two surfaces (e.g., beads, cover slips or cantilevers) to enable their manipulation and detection. This leads to one of the major challenges in single-molecule experimentation—verifying that exactly one molecular tether is being pulled, and distinguishing this tether from non-specific and unintended interactions that may occur (e.g., surface-surface interactions, formation of multiple bonds). The success and reliability of single-molecule experiments depends upon the creation of reliable, verifiable and robust linking techniques. This is particularly important for bond rupture studies (e.g., characterizing the strength of molecular adhesion bonds (Evans E. et al. (1997) Biophys. J. 72: 1541-55), DNA base pairing (Strunz T. et al. Proc. Natl Acad. Sci. 96: 11277), and cell adhesion and signaling (Evans E. A. et al. (2007) Science 316: 1148)), as the dissociation between two molecules can be difficult to positively identify due to the lack of an obvious mechanical signature.